Immunochemical studies of beta h-LPH. Antiserum to human beta-lipotropin has been raised in rabbits and characterized by gel double diffusion, quantitative precipitin, micro complement fixation and biological neutralization tests. A sensitive radioimmunoassay for human beta-lipotropin has been developed. It was demonstrated that the antiserum shows specificity for this hormone. Human beta-melanotropin and human beta-endorphin showed only a minimal degree of crossreaction, while ovine beta-lipotropin showed a partial but significant degree of cross-reactivity. The synthesis of alpha h-ACTH, (3,5-I2Tyr2,)-alpha h-ACTH, (3,5-I2Tyr23)-alpha h-ACTH, and (3,5-I2Ty42,23) alpha-ACTH was achieved by the solid-phase method. 3,5-Diidotyrosine was introduced as its Na alpha-Boc-O-(3-bromobenzyl) derivative. The completed peptides were purified by gel filtration on Sephadex G-10, chromatography on CMC, partition chromatography on Sephadex G-50, and gel filtration on Sephadex G-25. The final products were each characterized and found to be homogeneous by seven different analytical criteria. The steroidogenic activity of alpha h-ACTH was much more dependent on the integrity of Tyr2 than on that of Tyr23. Three analogs of beta-EP have been synthesized by the solid-phase method: beta c-EP-(1-5)-(28-31), beta c-EP-(6-31) and beta h-EP-(1-5)-(16-31). The analgesic activities of these synthetic peptides relative to that of the parent molecule show that all three peptides at high doses exhibit either no or much weaker analgesic activity than beta-EP. These data suggest that the entire beta-EP molecule is necessary for full in vivo analgesic activity.